Physiology and Glomerular Projections of Olfactory Receptor Neurons On

N. K. Hillier Æ C. Kleineidam Æ N. J. Vickers Physiology and glomerular projections of olfactory receptor neurons on the antenna of female Heliothis virescens (Lepidoptera: Noctuidae) responsive to behaviorally relevant odors

Abstract The neurophysiology and antennal lobe pro- tive to male hairpencil components and plant volatiles jections of olfactory receptor neurons housed within were also localized to distinct glomerular locations. This short trichoid sensilla of female Heliothis virescens information provides insight into the representation of F. (Noctuidae: Lepidoptera) were investigated using behaviorally relevant odorants in the female moth a combination of cut-sensillum recording and cobalt- olfactory system. lysine staining techniques. Behaviorally relevant odorants, including intra- and inter-sexual pheromonal Keywords Olfaction Æ Cobalt–lysine staining Æ compounds, plant and floral volatiles were selected for Sensillum Æ Antennal lobe Æ Receptor neuron testing sensillar responses. A total of 184 sensilla were categorized into 25 possible sensillar types based on Abbreviations Z11-16:Ald: (Z)-11-hexadecenal Æ odor responses and sensitivity. Sensilla exhibited both Z9-14:Ald: (Z)-9-tetradecenal Æ Z9-16:Ald: (Z)-9- narrow (responding to few odors) and broad (respond- hexadecenal Æ Z11-16:OAc: (Z)-11-hexadecenyl ing to many odors) response spectra. Sixty-six percent of acetate Æ Z11-16:OH: (Z)-11-hexadecen-1-ol Æ 14:OOH: the sensilla identified were stimulated by conspecific tetradecanoic acid Æ 16:OH: hexadecanol Æ 18:OH: odors; in particular, major components of the male octadecanol Æ 16:OAc: hexadecanyl acetate Æ 18:OAc: H. virescens hairpencil pheromone (hexadecanyl acetate octadecanyl acetate Æ 16:OOH: hexadecanoic and octadecanyl acetate) and a minor component of the acid Æ 18:OOH: octadecanoic acid Æ Z3-6:OH: (Z)-3- female sex pheromone, (Z)-9-tetradecenal. Following hexen-1-ol Æ Z3-6:OAc: (Z)-3-hexenyl acetate Æ characterization of the responses, olfactory receptor E2-6:Ald: (E)-2-hexenal Æ ORN: olfactory receptor neurons within individual sensilla were stained with co- neuron Æ PN: projection neuron Æ AL: antennal balt lysine (N=39) and traced to individual glomeruli in lobe Æ MGC: macroglomerular complex Æ LFG: large the antennal lobe. Olfactory receptor neurons with female glomeruli Æ cLFG: central large female specific responses to (Z)-9-tetradecenal, a female H. vi- glomerulus Æ mLFG: medial large female rescens sex pheromone component, projected to the fe- glomerulus Æ EAG: electroantennogram Æ SSR: single male-specific central large female glomerulus (cLFG) sensillum recording and other glomeruli. Terminal arborizations from sensillar types containing olfactory receptor neurons sensi- Introduction

Electronic Supplementary Material Supplementary material is In male and female moths, odors play an important role available for this article at http://dx.doi.org/10.1007/s00359 005 0061 x and is accessible for authorized users. in the location of potential mates, food sources and oviposition sites (Carde´ 1984; Hartlieb et al. 1999; N. K. Hillier (&) Æ N. J. Vickers Kennedy 1977; Landolt and Phillips 1997; Visser 1986). Department of Biology, University of Utah, In particular, the orientation of male moths to female Room 201 South Biology, Salt Lake City, UT 84112, USA E mail: [email protected] produced sex pheromone is a well-described phenome- Tel.: +1 801 5814795 non (Carde´ and Baker 1984; Kennedy and Marsh 1974; Fax: +1 801 5814668 Kennedy et al. 1980; Kennedy et al. 1981; Murlis et al. 1992; Vickers and Baker 1992). The behavioral, sensory C. Kleineidam Department of Behavioral Physiology and Sociobiology, and central discrimination of intra- and inter-speciﬁc Zoologie II, Biozentrum, Am Hubland, odor cues by male moths while locating a mate have 97074, Wu¨ rzburg Germany been a focal point of research on insect olfaction 200

(Hildebrand and Shepherd 1997; Roelofs 1979; Vickers The ORNs project to the antennal lobe (AL), the et al. 1998). Much less effort has been devoted to the primary olfactory neuropil, and make synaptic connec- study of olfactory processing in female moths. tions with local interneurons and central projection Nevertheless, many behavioral studies have revealed neurons (PNs; Hansson 1997; Mustaparta 2002). The that odors play an important role in the life history of insect AL is organized into discrete glomeruli that are adult female moths. Several studies have recognized the specialized for perception of particular odors (Hansson importance of host plant odor in female oviposition site et al. 1992; Hansson and Christensen 1999; Hildebrand choice (Reviews in Bernays and Chapman 1994; Carde´ 1996; Hildebrand and Shepherd 1997; Vickers and 1984; Finch 1986; Murlis 1997; Murlis et al. 1992; Visser Christensen 2003). In all moth species investigated to 1986). Female Heliothis virescens F. and Heliothis sub- date, there is a specialized cluster of sexually dimorphic, flexa Guene` e have been shown to engage in upwind male-specific glomeruli at the base of the antenna, flight toward several different plant volatiles (Mitchell known as the macroglomerular complex, or MGC (Berg et al. 1991; Tingle and Mitchell 1992; Tingle et al. 1989). et al. 1998; Christensen 1997; Christensen and Hild- DeMoraes et al. (2001) demonstrated that nocturnal brand 1987; Hansson et al. 1992, Todd et al. 1995; emissions from host plants under attack by H. virescens Vickers et al. 1998). The glomerular projections of larvae were repellant to conspecific females. Female pheromone-receptive ORNs have been traced to the moths, therefore, use olfactory cues as an important male MGC of H. virescens using cobalt-lysine stains, element of host plant choice. Additionally, floral odors linking peripheral physiological responses to individual may be important for both location of adult food glomeruli in the moth AL (Berg et al. 1998; Hansson sources (nectar) and in evaluation of host plant quality et al. 1995). Furthermore, the structure and physiology (Bruce et al. 2002; Cunningham et al. 2004; Raguso and of the MGC has been investigated through central Willis 2002). Helicoverpa armigera females were at- recording of PNs in the AL and found to be organized tracted to traps baited with floral odor lures, and elec- differently between closely related heliothine species troantennogram responses were elicited by exposure to (Christensen et al. 1991, 1995; Vickers and Christensen floral odors (Bruce and Cork 2001). 2003; Vickers et al. 1998). The organization of the MGC In addition to plant volatiles, female moths may is specialized for the processing of attractive and repel- detect a multitude of additional odorants, including lent odors in the pheromone blends of closely related volatiles associated with conspecifics (Birch 1975; Birch species, thus preventing cross-attraction and mating et al. 1990; Fitzpatrick and McNeil 1988; Ochieng’ mistakes (Klun et al. 1979, 1980a, b, 1982; Quero and et al. 1995). In H. virescens, male-produced hairpencil Baker 1999; Quero et al. 2001; Teal et al. 1981, 1986; compounds are important in courtship, wherein Tumlinson et al. 1975; Vickers 2002). females may reject males lacking the appropriate In female H. virescens, two female-specific glomeruli, complement of odors (Hillier and Vickers 2004). Fur- called the large female glomeruli (LFGs) are present at thermore, in several species of insects, females may the base of the antennal nerve (Berg et al. 2002). While have receptors that respond to odors found in their the physiology of the neurons associated with these own pheromone blend (den Otter et al. 1978; Hansson glomeruli is not known in H. virescens, studies of LFG et al. 1989; Ochieng’ et al. 1995; Seabrook et al. 1987). PNs in Manduca sexta L. have demonstrated that these Females of Spodoptera littoralis have sensilla that glomeruli may be specialized for host odor processing respond to a component of the female-produced sex (King et al. 2000; Shields and Hildebrand 2001). The pheromone (Z)-9, (E)-11-tetradecenyl acetate (Ljung- function and physiology of the remaining glomeruli that berg et al. 1993), while female Choristoneura fumiferana appear to be common to both the male and female AL is Clemens exhibit behavioral and electrophysiological less understood, and few studies have documented responses to (E)-11-tetradecenal and (Z)-11-tetradecenal, physiological activity directly within the female AL components of the female-produced sex pheromone (Anton and Hansson 1994; Carlsson et al. 2002; Mas- (Palaniswamy and Seabrook 1978). In these instances, ante-Roca et al. 2002; Meijerink et al. 2003). Galizia it is not clear whether these receptors are specialized et al. (2000) found calcium-imaging responses evoked by for pheromone reception or serve an alternative func- several plant odors that were localized to similar regions tion and are coincidentally activated by pheromone of the AL in H. virescens males and females. In a more components. recent study, Skiri et al. (2004) used calcium imaging to Male heliothine moths are considered more sensitive record spatio-temporal responses to selected plant odors than females to female-produced pheromones, having a in the ALs of male and female H. virescens. These population of sexually dimorphic long trichoid sensilla studies provide some indication of the possible sites for specialized for reception of sex pheromones (Almaas odor processing but were limited by the ability of cal- and Mustaparta 1990; Almaas et al. 1991; Baker et al. cium imaging to penetrate the AL and resolve activity in 2004; Cosse´ et al. 1998; Ochieng’ et al. 2003). In addi- glomeruli beneath the AL surface. tion to the long trichoid sensilla, there is a complement The aim of this study was to determine the response of short trichoid sensilla present on both sexes that is spectrum of ORNs within sensilla on female H. virescens thought to house olfactory receptor neurons (ORNs) antennae to a variety of behaviorally relevant odorants. sensitive to plant odors (Almaas and Mustaparta 1991). Additionally, we sought to determine the glomerular 201 projections of ORNs, to gain further insight into the 25C, 60% relative humidity, and set on a reversed light processing of odors in the female AL. Through this, we schedule (14L:10D) until eclosion. hope to provide a foundation for investigating female olfactory physiology, and permit comparison of olfactory processing between male and female insects. Chemicals

Chemicals were selected for female responses based on Materials and methods previously known olfactory stimuli implicated in behavioral studies on male and female H. virescens and Insects other heliothines (Table 1; Bruce and Cork 2001;De Moraes et al. 2001; Hillier and Vickers 2004; Teal and Three to four-day-old adult male and female Heliothis Tumlinson 1989). Stimuli selected were known compo- virescens were used from the colony at the University of nents of the female sex pheromone, compounds isolated Utah. Larvae were reared on a pinto bean diet (Shorey from male hairpencil extractions, ﬂoral volatiles and and Hale 1965) and sexes separated upon pupation. induced plant volatiles. Solutions of female sex phero- Pupae were placed in an environmentally controlled mone components (Z)-11-hexadecenal (Z11-16:Ald), chamber (Percival Scientiﬁc, Boone, Iowa, USA) at (Z)-9-tetradecenal (Z9-14:Ald), (Z)-9-hexadecenal

Table 1 Behavioral relevance for selected odorants for physiological activity in female Heliothis virescens

Odorant Behavioral role Citations

Pheromone components Z11 16:Ald Major component of H. virescens Cork et al. (1992); Cork and Lobos (2003); female sex pheromone, Klun et al. (1979); Klun et al. (1980a, b); male H. virescens attraction. Teal et al. (1984); Teal et al. (1986); Z9 14:Ald Minor component of H. virescens Tumlinson et al. (1975); Vetter female sex pheromone, and Baker (1983); Vickers (2002) male H. virescens attraction. Z11 16:OAc Minor component of H. subflexa female sex pheromone, male H. virescens antagonist. Z11 16:OH Present in small amounts in the H. virescens female sex pheromone gland, behaviorally important in other Heliothine species (H. zea and H. subflexa). Z9 16:Ald Behaviorally important in other Heliothine species (H. zea and H. subflexa; major component in H. assulta). Hairpencil components 16:OAc Compounds present in male H. virescens Hillier and Vickers (2002); 18:OAc male hairpencils. Presented as a blend Teal and Tumlinson (1989) 16:OH during courtship, they produce female 18:OH quiescence and mate acceptance. 16:OOH 18:OOH Floral odorants Benzyl alcohol Odorants elicited EAG responses Bruce and Cork (2001); Bruce et al. (2002); Benzaldehyde in female Helicoverpa armigera. Cunningham et al. (2004); Hartlieb Phenyl acetaldehyde Females engaged in upwind flight and Anderson (1999); Hartlieb Phenyl acetylene and attraction to floral baits, and Rembold (1996). 2 Phenyl ethanol and could be conditioned to floral odors. Induced plant volatiles a humulene Nocturnal volatiles emitted by DeMoraes et al. (1999); (Z) 3 hexenol host tobacco plants, Nicotiana tabacum, DeMoraes et al. (2001); (E) 2 hexenol during H. virescens larval feeding Dickens et al. (1990). Linalool are highly repellent to female moths b caryophyllene and attractive to parasitoids, (Z) 3 hexenyl acetate and can increase attraction of male H. zea (Z3 hexenyl acetate) when co emitted with sex pheromone. 202

(Z9-16:Ald), (Z)-11-hexadecenyl acetate (Z11-16:OAc) separated by 1 s each. Two seconds of pre-stimulation and (Z)-11-hexadecenol (Z11-16:OH)) were obtained and 1 s of post-stimulation data were recorded, resulting from Bedoukian Research Inc (Danbury, CT, USA). in six seconds total recording time for each stimulus Solutions of hairpencil components (tetradecanoic acid presentation. Spike frequency was determined by (14:OOH), hexadecanol (16:OH), hexadecanyl acetate counting the number of spikes during 1 s following each (16:OAc), hexadecanoic acid (16:OOH), octadecanol stimulus onset and standardized by subtracting the (18:OH), octadecanyl acetate (18:OAc) and octadeca- number of spikes during the first second of pre-stimulus noic acid (18:OOH)) were obtained from either Dr. recording time. For calculation of dose-response curves, James Tumlinson (Department of Entomology, The the mean number of spikes across all the three stimuli Pennsylvania State University, University Park, PA, was used (Main-effects ANOVA indicated no significant USA), or purchased from Sigma Aldrich (St. Louis, difference in the spike frequency of three consecutive MO, USA). Solutions containing floral odorants (benzyl stimuli). alcohol, 2-phenyl ethanol, benzaldehyde, phenyl acetaldehyde and phenyl acetylene) and plant volatile (linalool, (Z)-3-hexenyl acetate (Z3-6:OAc), (Z)-3-hexenol Electroantennogram recordings (Z3-6:OH), (E)-2-hexenal (E2-6:Ald), b-caryophyllene and a-humulene) solutions were kindly provided by Dr. Electroantennograms were conducted on isolated Robert Raguso (Department of Biological Sciences, antennae of both male and female H. virescens (Roelofs University of South Carolina, Columbia, SC, USA). All 1979; Schneider 1962). Antennae were excised at the odorant solutions were stored in a freezer at À20C. scape using iridectomy scissors. The tip was cut at the Samples of stock solutions were confirmed to be at distal 4–5th segment. Chloridized silver electrodes were >95% purity by injection onto a Shimadzu GC 17A gas inserted into the tip and the base of the antenna, and chromatograph equipped with a 30 m · 0.25 mm ID sealed with vaseline. DB-5 capillary column. All chemicals were diluted in The EAG activity was filtered (HUMBUG, Quest hexane at decade steps from 100 ng to 1 mg, with the Scientific), amplified 1,000· (ER-1, Cygnus Technol- exception of 16:OH, 18:OH, 16:OOH and 18:OOH, ogy), monitored on an oscilloscope (GOS-620FG, which all became solid at room temperature at concen- Instek) and data recorded using a Rec-Wise software trations higher than 100 lg/ll. program (written by C.K.) in Labview 6.1. The integration above each EAG curve was recorded, and standardized by subtracting the consecutive hexane Odorant stimulation blank in a series. The EAG integration values were normalized between males and females by expressing all Stimulus cartridges were made by applying 10 llofan responses as a percentage of the mean response to a odorant solution to a 5·30 mm2 piece of filter paper in a 100 ng linalool stimulus for each sex (set at 100%). EAG 1 ml plastic syringe. Stimulus loadings ranged from responses were compared using a nested ANOVA 100 ng to 100 lg for EAGs, and from 100 ng to 1 mg comparing sex, odor and concentration effects. Means for single sensillum recording (SSR). Stimulation was were separated using a Fisher’s Least Significant Dif- automatically controlled by Labview 6.1 software ference test (P<0.05). Statistical analyses were con- (National Instruments, Austin, TX, USA). A continu- ducted using Statistica (StatSoft Inc., 1999). ous flow of charcoal-filtered, humidified air was provided at a flow rate of 1 l/min. To activate stimulation, a valve driver (Parker-Hannafin) was used to switch the Single sensillum recordings (SSRs) airstream from the continuous flow to the stimulus cartridge. Both the stimulus and continuous flow were Electrophysiological responses of ORNs in individual connected to a mixing chamber (50 mm long · 5mm sensilla were conducted using a cut sensillum technique inner diameter), with 20 mm of thin plastic straws in- (Kaissling 1974; Van der Pers and den Otter 1978). serted at the exit to smooth the flow exiting the chamber. Individual moths were restrained in 1 ml cut disposable The exit of the mixing chamber was positioned 10 mm pipette tips, and their heads held in place with dental away from the insect antenna. For EAGs, individual wax. The moths were placed horizontally on a depres- stimuli were presented in increasing concentration steps, sion slide, and the tip of the antenna was fastened to the beginning with 100 ng and ending with 100 lg, and the side of the slide using water-soluble correction fluid order of the individual stimulus presentation was ran- (Liquid Paper, Paper Mate). Once the moth was se- domized. For SSRs, stimuli were presented in random cured, a silver chloride reference electrode was inserted order, and at a concentration of 100 lg for initial into the contra-lateral eye. screening of a sensillum, with at least 60 s between Prepared moths were mounted on a compound stimulation to prevent adaptation, and ensure that spike microscope (Wild) and viewed at 20·. The rig was activity had returned to pre-stimulation levels. Between mounted on an anti-vibration table, and shielded with a all stimulus presentations, a hexane blank was tested. Faraday cage. Sensilla were cut using a thin glass cap- Stimulation occurred as a series of three 100 ms puffs, illary mounted to a piezo crystal, which was connected 203 to a function generator. The glass capillary resonated at (100 ms ‘puff’ every 2 s) and the electrode was allowed high speed by altering the frequency and amplitude of to remain in contact with the sensillum for a total of 1 h the signal from the function generator to the sound- (in cases where several odorants activated the sensillum, emitting piezo as described by Go¨ dde (1989). This tool the odor to which the neuron responded most sensitively was then used to cut the distal 20% of a sensillum by was selected for stimulation). After 1 h, the moth was advancing the vibrating glass capillary on a microma- removed to a petri dish with moistened paper toweling, nipulator. Sensilla were randomly selected from the and placed in a 4C refrigerator for 48 h. The brain was proximal ventral surface of the antenna. Once cut, the dissected from the moth and placed in a saline solution cutting tool was removed from the micromanipulator with 3–6 drops of ammonium sulfide on a rotator for and replaced with a headstage recording electrode, and 10 min. The brain was rinsed 3–4 times in saline, and was placed over a single sensillum. The recording elec- fixed overnight in AAF [100% Alcohol: Acetic acid: trode consisted of a chloridized silver wire in a saline- 38% Formaldehyde (8:1:2)] fixative. The next day, filled glass capillary. brains were rinsed three times in 96% ethanol, and Previous research has shown that only a single mor- stored in 70% ethanol in the refrigerator until silver phological subtype of sensilla trichodea (length 30– intensification procedures could be performed. 50 lm and diameter 2 lm) is present on the antenna of Brains were subjected to Timms silver intensification female H. virescens, thus preventing any confusion be- (Timm 1958) for 20–30 min. Following intensification, tween morphologically distinct sensillar subtypes brains were serial dehydrated in ethanol and embedded (Almaas and Mustaparta 1990). This is also the case in in Spurrs resin. Embedded brains were sectioned at Helicoverpa assulta, wherein only a single morphological 10 lm, and placed on subbed slides. Slides were count- subtype of s. trichodea has been described in females erstained using a modified solution of Lee’s methylene (Koh et al. 1995). Moreover, the length and diameter of blue-basic fuchsin solution (methylene blue +Azure II these s. trichodea made them easy to discriminate from in borate):0.5% Basic Fuchsin in 95% ethanol:100% other sensilla such as larger sensilla chaetica or much ethanol = 1:2:1 (S.G. Lee, personal communication). shorter sensilla basiconica (<10 lm length). Slides were coverslipped and observed at 20–40· on a Following presentation of the complement of odor- compound microscope with an Optronics Microfire ants, if the signal was sufficiently strong, responding digital camera attached. ORNs were tested again to obtain dose–response curves for stimuli. Odors were presented from low to high Digital reconstructions concentrations from 100 ng/ll to 1 mg/ll (except for previously noted compounds which solidified at con- Digital images of serial sections were taken at 20· to centrations greater than 100 lg/ll). ORN activity was provide the branching pattern and glomerular projec- filtered and amplified (1,000 ·) using similar equipment, tions of the stained ORNs, and perform reconstruc- software and protocols as in EAG experiments. In in- tions of the AL. Reconstructions were made in a stances where it was suspected more than one ORN ‘Single Blind’ format, wherein the brains were coded as might be present, spike amplitudes were also examined. image files and reconstructed without knowledge of Filtering, spike detection and analysis were also per- their ORN responses. Digital images were saved as formed using programs written by C. K. on Labview TIFF files and imported to AMIRA 2.3 (Indeed 6.1. A main effects ANOVA was used to determine GmbH, Berlin; http://www.amiravis.com) for recon- significant differences in spike frequency based on struction. Individual glomerular boundaries and the odorant, concentration or stimulus number (1, 2 or 3), outline of each of the whole AL from each section and means were separated using Fisher’s LSD test. were traced with an image segmentation tool that permitted 3D grouping of labeled materials for each glomerulus. Reconstructed ALs and stained ORN Staining of sensory neurons projections were compared visually against previously constructed models of female ALs and identified using Once the physiology of ORN(s) within a sensillum was the H. virescens AL atlas (Berg et al. 2002). Images characterized, attempts were made to stain any neu- and digital reconstructions were exported to Adobe ron(s) through a cobalt-lysine staining procedure Photoshop 6.0, labeled and adjusted for brightness and (Hansson et al. 1995; Todd et al. 1995; Todd and Baker contrast where necessary. 1996). For sensilla selected for staining with cobalt- lysine solution, the saline-filled recording electrode was replaced with an electrode containing 0.5 M solution of Results cobalt lysine (2.38 g cobaltous chloride with 5 g L-lysine in 20 ml of distilled water, lowered to a pH of 7.2–7.4 Electroantennogram recordings using concentrated HCl). Preparation of this solution has been described previously (Ochieng’ et al. 1995; Comparison of electroantennogram responses between Hansson et al. 1995; Todd et al. 1995). ORNs were male and female H.virescens indicated distinct differ- stimulated with an appropriate odorant for 10 min ences between sexes for many of the odorants tested 204

Fig. 1 Electroantennogram (EAG) responses from male (N=10) Z9 14:Ald at a ratio of 90:10, concentration represents loading of and female (N=10) Heliothis virescens. All responses were Z11 16:Ald); b Floral volatiles; c Male H. virescens hairpencil standardized to a hexane blank before analysis, and subsequently components; and d Induced plant volatiles.Asterisks indicate normalized as a percentage of a response to 100 ng linalool signiﬁcant diﬀerences between mean male and female EAG stimulus (measured as 100%) for each sex. Responses to: a Female responses (P<0.05, Fisher’s LSD) H. virescens sex pheromone components (blend of Z11 16:Ald and

(Fig. 1a–d). Concentration-dependent increases in EAG viable ORNs present in 955 sensilla trichodea. Of those response were found in both sexes to most odorants, yet sensilla screened, 184 exhibited clear responses to the the magnitude of the response to different odors varied odorants tested, with 25 possible ‘sensillar types’ iden- between the sexes (F1,18= 4.1, P<0.001). Male re- tified based upon response profiles of ORNs therein sponses to female sex pheromone components Z11- (Table 2). Twenty out of the 22 odorants tested elicited 16:Ald and Z9-14:Ald were significantly greater than responses from ORNs in one or more sensillar types. female responses; however, females also showed re- Surprisingly, 16:OOH and 18:OOH, both found in male sponses to both compounds, particularly Z9-14:Ald. H. virescens hairpencil extracts did not elicit responses Several hairpencil components produced larger EAG from ORNs in any of the sensilla contacted. There was responses in females than in males, notably: 16:OAc, no specialization of sensillar types noted by the position 16:OH, 18:OAc and 18:OH. Female responses were on the antenna. significantly greater than males for benzaldehyde, benzyl Many of the 25 sensillar types could be grouped alcohol, 2-phenyl ethanol and phenyl acetaldehyde. Fe- based on ORN responses to four ‘categories’ of odors male EAG responses were also significantly higher than tested (hairpencils, female sex pheromones, florals and male responses when stimulated with plant volatiles induced plant volatiles; Tables. 1, 2). Two sensillar types known to be induced by feeding damage, particularly housed ORNs responding to male hairpencil compo- linalool and Z3-6:OH. nents, four to female heliothine sex pheromone components, three to floral plant odors and seven to induced plant volatiles (Table 2). Four sensillar types contained Single sensillum recordings ORNs that responded to a combination of floral and plant volatiles, and five other sensillar types were found Single sensillum recordings were made from 157 female in which ORNs exhibited broad responses to several H. virescens antenna with good electrical contact and different types of odor. 205

Table 2 Response proﬁles for 25 sensillar types identiﬁed from 157 H.virescens females through cut sensillum recording

Responses are indicated by asterisks in the boxes for corresponding odorants to the sensillar type, with *=5 10 spikes sÀ1, **=10 20 spikes sÀ1 and ***=20 or more spikes sÀ1 following a 100 ms pulse of 100 lg of stimulus. Responses represent the relative increase in spiking above a threshold of spontaneous activity as standardized by a hexane control stimulus

Types 1, 3 and 4 (with ORNs responding to hair- Successful stains, wherein axonal projections could be pencil and pheromone components) were the most visualized to their terminal arborizations within the abundant of the sensilla identified, revealing that 66% of individual glomeruli, were found in 39 of the 102 at- the total sensilla contacted contained ORNs sensitive to tempts, with stain present in 18 additional brains with- conspecific odors. In most (N=17) of the sensillar types out clear arborizations. ORNs from seven different identified, ORN responses were specific to four or fewer sensillar types were stained (Tables. 2 and 3), with suc- compounds within a given category. The remaining nine cessful stains primarily from type 1, 3 and 4 sensilla. sensillar types (17–25) contained ORNs which were Nineteen stains revealed uniglomerular axonal projec- broadly tuned to a large number of odorants (3–18 tions while 20 contained multiple ORNs projecting into odorants). different regions of the AL (Figs. 2, 3, 4, 5, 6, 8, 9). No The intensity of ORN background firing varied be- stains were evident in tissues beyond the AL. tween sensillar types. Most ORNs exhibited background firing rates of 1–3 spikes sÀ1, with a few (<5%) showing background activity as high as 10–12 spikes sÀ1. Inhib- Sensillar types responding to pheromonal odorants: itory responses were not noted to any of the odors tested. All ORNs responded in a relatively phasic manner, The most common sensillar type, found in 29% of with some sensillar types having ORNs which main- responses, were type 1 sensilla (N=54), with an ORN tained tonic firing for several seconds following stimu- that responded specifically to the male hairpencil lation, particularly at high stimulus loadings (Figs. 2, 3, components 16:OAc and 18:OAc, and to Z11-16:OAc 4, 5). No significant effect (P<0.05) was found due to (Fig. 2a–e). These ORNs were sensitive to stimulus consecutive presentation of the same odorant in any of loadings between 1 lg–1 mg and responded to no the sensilla tested. other odorants. No differentiation could be made be- Most sensilla contained 1–2 active ORNs as deter- tween spike amplitudes for ORN responses in these mined by the separation of spike amplitudes, however, sensilla, so it is possible that activity was present in a the possibility exists that additional silent or spontane- single ORN, potentially responding to specific com- ously active ORNs may have been present which were pounds containing an acetate functional group (though not detected, or could not be distinguished based on the no responses were noted to Z3-6:OAc). In a few in- spike amplitude. Unless otherwise noted, all spikes re- stances (N=5), a second, small spike amplitude ORN corded from ORNs in a sensillar type were of similar was present which did not respond to odor stimulation amplitude. (not shown). 206

Fig. 2 Type 1 sensillum ORN responses, stain and 3D reconstruc 3·100 ms pulses. f g Cobalt lysine stain and 3D reconstruction tion. a d ORN response proﬁle (original spike trains) from a of an ORN glomerular projection from a type 1 sensillum. female H.virescens type 1 sensillum, responding primarily to male Uniglomerular arborization in glomerulus 59, located antero produced hairpencil components: a Z11 16:OAc, b 18:OAc, c laterally near the base of the antennal nerve (N=6). Arrowheads 16:OAc and d hexane blank. e Dose response curves from ORNs in indicate location of stain within micrograph. Dorsal D; Medial M. type 1 sensilla (N=15) stimulated with 16:OAc, 18:OAc and Z11 Scale bars =100 lm 16:OAc. Six second total recording time, stimulus delivery

Type 1 sensilla showed an ORN with consistent 59 and the fourth to 2, 11, 19, 31, 59 and 60 (Table 3). In arborization in glomerulus 59, anterior to the cLFG the latter case, there may have been leakage at the (Fig. 2). In 34 staining attempts, 14 successful stains electrode tip, causing adjacent sensilla to become loaded were made. In six preparations, uniglomerular projec- with cobalt lysine, and thus, non-informative glomeruli tions were noted to glomerulus 59 (Fig. 2f–g). Three may have been stained. All type 1 stains included a preparations resulted in a pair of ORNs being stained projection to glomerulus 59. and projecting to glomeruli 40 and 59 (Table 3). In two Seven type 2 sensilla were identiﬁed with ORNs other preparations, double stains were also made to a which responded exclusively to 16:OH, a component of pair of glomeruli: 48 and 59 (N=1) and also to glome- the male hairpencil pheromone (Fig. 3a–c). A second ruli 57 and 59 (N=1; Table 3). Four other stains showed non-responsive ORN was also evident in all recordings multiple projections, the ﬁrst to glomeruli 3, 10 and 59, from type 2 sensilla. In six attempts, three ORNs were the second to 8, 36 and 59, the third to glomeruli 48 and stained which were excited by 16:OH alone. All of the 207

Table 3 Summary of projections identiﬁed from ORNs in sensillar types stained in female H.virescens

Sensillar type Physiology N Glomerular projections

Hairpencil components Type 1 Z11 16:OAc, 16:OAc, 18:OAc 6 59 3 40, 59 1 48, 59 1 57, 59 1 3, 10, 59 1 8, 36, 59 1a 2, 11, 19, 31, 59, 60 Type 2 16:OH 3 41 Female sex pheromone components Type 3 Z9 14:Ald 5 cLFG 4 34, cLFG 1 40, cLFG 1 40, 51, cLFG Type 4 Z9 14:Ald, Z9 16:Ald 2 20 1 17, 20, cLFG 2 20, cLFG 1 38, cLFG Type 5 Z11 16:Ald, Z9 14:Ald (weak), 160 Z9 16:Ald (weak), Z11 16:OH (weak) Induced plant volatiles Type 12 Linalool 2 36, mLFG Type 13 Linalool,b caryophyllene, 26 Z3 hexenol, Z3 hexenyl acetate Total stains (from 102 attempts): 39

Glomerular positions indicated numerically from the H. virescens antennal lobe atlas (Berg et al. 2002). Consistent glomerular targets of axonal arborization are indicated in bold a Possibly more than one sensillum stained identified ORNs projected to glomerulus 41, located uniglomerular arbor to glomerulus 60, located anterior posterior to the cLFG (Fig. 3d, Table 3). to glomerulus 59 (Fig. 6g). Type 6 sensilla (N=3) con- Type 3 sensilla ORNs (N=45) were sensitive to Z9- tained ORNs which responded weakly to Z11-16:OH 14:Ald alone and showed no activity to other odorants (Table 2). (Fig. 4a–e). Eleven successful stains were made from ORNs in type 3 sensilla. Of these, five arborized only in cLFG, and the remainder showed multiglomerular Sensillar types responding to plant volatiles stains between cLFG and other glomeruli (34, 40, 51; Fig. 4f–g, Table 3). Several ORNs responded weakly to floral odorants, Type 4 sensilla ORNs (N=24) responded strongly to particularly in sensilla that responded to a wide range of Z9-14:Ald at concentrations between 1 lg and 1 mg, odors. ORN responses were noted to benzyl alcohol and and had a higher activity threshold for Z9-16:Ald, 2-phenyl ethanol in types 7 (N=2) and 8 (N=4) sensilla responding to concentrations between 100 lg and 1 mg (Fig. 7, Table 2). No differences in amplitude were evi- (Note, however, that Cosse´ et al. (1998) found that Z9- dent between floral responses by ORNs within these 14:Ald is emitted at a ten times greater rate than Z9- types of individual sensilla. Type 8 sensilla also housed a 16:Ald from similarly loaded cartridges. Therefore, non-responsive large amplitude spiking ORN. No ORN taking into consideration the emitted stimulus rather stains were obtained for sensilla responding to floral than the cartridge loadings, the response threshold to odorants. Attempted staining of type 9 sensilla was each of these odorants may be more similar; Fig. 5a–f). unsuccessful (N=1). Successful stains were made in 6 out of 14 attempts, with Sensillar type 11 (N=5) had small amplitude ORNs four different combinations of stained glomeruli. In that responded to Z3-6:OH and Z3-6:OAc (Table 2). these stains, some arborizations were either present Similar ORN(s) are evident in several other sensilla (for medially in glomerulus 20 or in cLFG, with additional example type 13), and ORNs in other sensillar types arborizations present in either glomerulus 17 or 38 responded weakly to either Z3-6:OH or Z3-6:OAc, (Fig. 5g–h, Table 3). possibly indicating differentially responding ORNs Three type 5 sensilla contained ORNs responsive which are common to several sensillar types. No ORN primarily to Z11-16:Ald, and slightly less sensitive to Z9- stains were obtained for type 11 sensilla. 14:Ald, Z9-16:Ald and Z11-16:OH, principally at con- Type 12 (N=8) and 13 (N=8) sensilla both had centration loadings greater than 100 lg (Fig. 6a–f). A ORNs with strong responses to linalool at concentra- single stain was made from a type 5 sensillum, showing a tions between 10 lg and 1 mg (Figs. 8, 9). Type 12 208

ORNs were only activated by linalool, whereas type 13 ORNs also had secondary responses to b-caryophyllene, Z3-6:OH and Z3-6:OAc (Figs. 8a–c, 9a–f). Two to three ORNs are evident in this sensillar type, one or more small spike amplitude ORN with responses to Z3-6:OH and Z3-6:OAc (Fig. 9c–d), a large spike amplitude ORN responding to linalool and b-caryophyllene and potentially a non-responsive ORN of similar amplitude. Two successful stains were made from ORNs in type 12 sensilla and projected to glomeruli mLFG and 36 (Fig. 8d–e). Two ORNs in type 13 sensilla were also stained projecting ventrally to glomerulus 6 (Fig. 9g). Attempted staining of ORNs in Type 14 (N=2) and Type 16 (N=3) sensilla was unsuccessful. Neurons responding to b-caryophyllene were noted in several sensillar types. Type 16 (N=5; Fig. 10a–c) sensilla contained ORNs which responded exclusively to b-caryophyllene, while type 14 (N=2) and 15 (N=2) showed weaker responses to a-humulene and Z3-6:OH or E2-6:Ald, respectively (Table 2). An additional small amplitude, non-responsive ORN was found only in type 15 sensilla. Responses to various combinations of ﬂoral and plant volatiles were noted in some ORNs (sensillar types 17–20), possibly indicating co-localization of various ORNs found in sensillar types 7–16 (Table 2). No clear stains were obtained from ﬂoral or plant volatile responding ORNs in sensillar types 17–20.

Broadly responding ORNs

Five different sensilla housed ORNs responsive to a range of odorants (‘‘Broadly tuned’’, Table 2). The ORN response spectra for most of these sensilla do not appear specialized for a particular functional group across the range of odorants tested, but exhibit portions of ORN response profiles found in other sensilla. For example, the response spectrum of type 19 encompasses the combined stimulus profiles for ORNs in sensillar types 7 and 13. Spike amplitudes were variable within all of the broad-spectrum sensilla, with typically two or more ORNs being excited by the complement of odors tested. Unfortunately, no successful stains were achieved from ORNs in these ‘generalist’ sensilla (types 21–25) which exhibited responses to odorants from several different categories.

Discussion

Fig. 3 Type 2 sensillum ORN responses and 3D reconstruction. a Single sensillum recording veriﬁed the presence of ORNs b ORN response proﬁle (original spike trains) from a female H.virescens type 2 sensillum to: a 16:OH and b hexane blank. c responding to all of the odorants tested, with the Dose response curves from ORNs in type 2 sensilla (N=2) exception of 16:OOH and 18:OOH. Previous work has stimulated with 16:OH. Six second total recording time, stimulus shown EAG activity to be an accurate means of esti- delivery 3·100 ms pulses. d 3D reconstruction of a cobalt lysine mating total ORN activation at the whole-antenna level, stain of an ORN from a type 2 sensillum showing a uniglomerular projection to glomerulus 41 (posterior lateral region of the AL; for a particular odorant (Dickens et al. 1993; Nagai N=3). Dorsal D; Medial M. Scale bar=100 lm 1983; Visser 1979). Assuming that EAG response largely 209

Fig. 4 Type 3 sensillum responses, stains and 3D reconstructions. type 2 sensilla. d e Uniglomerular projection to cLFG (central Large a b ORN response proﬁle (original spike trains) from a female Female Glomerulus) dorso laterally located at the base of the H. virescens type 3 sensillum to: a Z9 14:Ald and b hexane blank. antennal nerve (N=5). f g Example of a multiglomerular double c Dose response curves from ORNs in type 3 sensilla (N=19) stain indicating axons projecting to cLFG and the adjacent stimulated with Z9 14:Ald. Six second recording time, stimulus glomerulus 34 (N=4). Arrowheads indicate location of stain within delivery 3·100 ms pulses. d g Cobalt lysine stains and 3D recon micrographs. Dorsal D; Medial M. Scale bars=100 lm structions of ORN glomerular projections from Z9 14:Ald sensitive 210

Fig. 5 Type 4 sensillum responses, stain and 3D reconstruction. a reconstruction of a double stain showing ORN axonal projections e ORN responses (original spike trains) from a female H. virescens from a type 4 sensillum to cLFG (central large female glomerulus) type 4 sensillum to: a 100 lg Z9 14:Ald, b 500 lg Z9 14:Ald, c and medially located glomerulus 20 (N=2, g is at a focal depth 100 lg Z9 16:Ald, d 500 lg Z9 16:Ald and e hexane blank. f Dose displaying stain in glomerulus 20, 3D reconstruction also indicates response curves from ORNs in type 4 sensilla (N=12) stimulated location of concurrent stain in cLFG). Arrowheads indicate with Z9 14:Ald and Z9 16:Ald. Six second recording time, stimulus location of stain within micrograph. Dorsal D; Medial M. Scale delivery 3·100 ms pulses. g h Cobalt lysine stain and 3D bars=100 lm reflected a similar activation of the ORN population on present between the sexes. This result contrasts with both the male and female antenna, our results suggest previous work on H. armigera (Hartlieb and Rembold that there is a variation in the complement of ORNs 1996) and Agrotis segetum Denis and Schiffermu¨ ller 211 c Fig. 6 Type 5 sensillum responses and 3D reconstruction. a e ORN response profile (original spike trains) from a female H. virescens type 5 sensillum to: a Z11 16:Ald and with a lower response to: b Z9 14:Ald, c Z11 16:OH, and d Z9 16:Ald; e hexane blank. f Dose response curves from ORNs in type 5 sensilla (N=12) stimulated with female H. virescens female sex pheromone components. Six second recording time, stimulus delivery 3·100 ms pulses. g 3D reconstruction showing an ORN axonal projection from a type 5 sensillum to glomerulus 41 (near base of AN; N=1). Dorsal D; Medial M. Scale bars=100 lm

(Hansson et al. 1989) in which the male and female moths demonstrated similar EAG responses to plant- produced volatiles. Not surprisingly, Z11-16:Ald and Z9-14:Ald elicited much higher EAG responses in male H. virescens, which have a large population of ORNs specific for each of these odorants (Almaas and Mus- taparta, 1991; Baker et al. 2004). Based on EAG responses and the population of ORNs sampled in this study, some specialization of ORN populations on female antennae is likely. Males exhibited EAG responses to most of the odorants tested; however, the sensitivity to hairpencil components and plant volatiles was lower than that observed in female H. virescens. With the exception of specialized dimorphic glomeruli (MGC in males and LFGs in females), previous work has shown that there was a similarity between the structural position and number of glomeruli present, along with similar odor-evoked spatial response patterns observed using calcium-induced fluorescence to odorants tested in the male and female H. virescens (Berg et al. 2002; Skiri et al. 2004). The correspondence in AL structure between the male and female moths may suggest a functional homology in the glomeruli of both sexes, with variation in glomerular size being an indication of relative input from ORNs at the periphery (such as the large glomeruli of the MGC being due to the increased peripheral input of pheromone-sensitive ORNs). Both the variation in EAG responses and AL structure in H. virescens provide for the interesting prospect of investigating homology between the sexes in the complement of ORNs in short s. trichodea, and their projections to the glomerular array. In many cases, the ORN response was clearly specialized to a specific odorant. This concurs with Stran- den et al. (2003b) who found that a single sensillum in H. virescens females generally responded strongly to one or two odorants and weakly to others. Conversely, we also found that many sensilla also contained more general responses, with the presence of multiple ORNs conferring a more general response profile in some sensilla. Koh et al. (1995) have shown that as many as four ORNs may be co-localized in the same sensillum of H. assulta Guene´ e. Recordings were attempted from 955 sensilla, with 771 sensilla non-responsive to the complement of odors tested (clean recordings, with spontaneous firing, but no which were not sensitive to any of the odorants tested. odor-induced excitation). Additionally, some sensillar This indicates that there are likely to be many more types contained silent or spontaneously firing ORNs olfactory sensillar types present on the antenna than 212

Fig. 7 Type 7 and type 8 sensillum responses. a e ORN responses (original spike trains) from a female H. virescens type 7 sensillum to: a benzyl alcohol, b 2 phenyl ethanol and with a lower response to c benzaldehyde and d phenyl acetaldehyde; e hexane blank. f Dose response curves from ORNs in type 8 (N=4) sensilla responding only to 100 lgof ﬂoral odors benzyl alcohol and 2 phenyl ethanol. Six second recording time, stimulus delivery 3·100 ms pulses

documented in this study. Stranden et al. (2003a, b) and particular, ORNs sensitive to (-) germacrene D were Røstelien et al. (2000b) previously identiﬁed potential found in 80% of the sensilla tested (Røstelien et al. plant-sensitive ORNs on female H. virescens antenna. In 2000a). Additionally, Stranden et al. (2003a, 2003b) 213

Fig. 8 Type 12 sensillum responses, stain and 3D reconstruction. showing a double stain from a linalool sensitive type 12 sensillum, a b ORN responses (original spike trains) from a female H. projecting to mLFG (medial large female glomerulus) and virescens type 12 sensillum to: a linalool; b hexane blank. c Dose glomerulus 36 (both near the base of the AN; N=2). Arrowheads response curves from ORNs in type 12 sensilla (N=4) stimulated indicate location of stain within micrograph. Dorsal D; Medial M. with linalool. Six second recording time, stimulus delivery Scale bars=100 lm 3·100 ms pulses. d e Cobalt lysine stain and 3D reconstruction found ORNs which were sensitive to several sesquit- sensillum is likely to contain a single ORN (as evidenced erpenes, E-b-ocimene, E,E-a-farnesene, homo-farnesene by several uniglomerular projections to glomerulus 59), and geraniol; co-localized within a sensillar type which responds to 16:OAc, 18:OAc and Z11-16:OAc. shared between three heliothine species. However, the This result indicates that the sensillum may be broadly behavioral relevance of these odorants has not been tuned to 16–18 chain acetates, or is specifically tuned to demonstrated in female H. virescens. Nevertheless, the these hairpencil compounds (no response was found to non-responsive sensilla in the current study may have the shorter chain Z3-6:OAc). Our previous work has housed ORNs sensitive to other odorants, including showed that when presented as individual compounds, those mentioned above, which were not utilized during 18:OAc appeared repellent to female H. virescens, while the course of our experiments. More recently Røstelien 16:OAc caused abdominal extension (Hillier and Vickers et al. (2005) published descriptions of 14 ORN 2004). This study also found that the blend quality was types sensitive to plant odors from H. virescens and important in mate choice, and therefore, combinations H. armigera, many responsive to odorants not tested in of odorants (and ORN activation) are probably signifi- our current study. cant in blend discrimination. Perhaps ORNs housed in We have previously demonstrated that male hair- other sensilla may act in concert with the type 1 sensilla, pencil odorants are important in mate selection by fe- thereby facilitating discrimination between the 16- and male H. virescens (Hillier and Vickers 2004). Constituent 18-chain acetate. components of these hairpencil blends include 16:OAc, Type 1 sensilla, at 29%, were the most abundant 18:OAc and 16:OH (Teal and Tumlinson 1986); odor- sensillar type. In fact, 74% of the sensilla sampled were ants for which we have now identified specific ORNs. tuned exclusively to conspecific odorants, including Additionally, it is interesting to note that the type 1 hairpencil odorants and female-produced sex pheromone 214

Fig. 9 Type 13 sensillum responses and 3D reconstruction. a e odorants. Six second recording time, stimulus delivery 3·100 ms ORN responses (original spike trains) from a female H. virescens pulses. g 3D reconstruction showing a uniglomerular projection to type 13 sensillum to: a linalool, b b caryophyllene, c Z3 hexenyl glomerulus 6 (antero ventral) from a type 13 sensillum (N=2). acetate, d Z3 hexenol and e hexane blank. f Dose response curves Dorsal D; Medial M. Scale bar=100 lm from ORNs in type 13 sensilla (N=4) stimulated with plant volatile components. EAGs from H. virescens females have also nerve in S. littoralis. Additionally, Stranden et al. previously shown responses to many components of the (2003a, b) reported stains of two sensillar types (only a female sex pheromone (Almaas and Mustaparta 1991). single stain from each study) responding to volatiles The presence of such an array of pheromone-sensitive from female H. assulta using tetramethylrhodamine receptors is surprising, given previous assumptions that dextran applied with a tungsten electrode. To our short trichoid sensilla are primarily used for detecting knowledge, the present study represents the first attempt non-pheromonal odors. Hansson et al. (1989) found that to identify a range of non-female sex pheromone- in the noctuid Agrotis segetum, female sensilla showed no receptive ORN projections in H. virescens. response to the major female-produced sex pheromone Much akin to the male MGC, staining of ORNs and components or their analogues (males of this species lack digital reconstructions indicate that many of the ORNs dimorphic pheromone-sensitive long trichoid sensilla). specialized for intra-specific odor processing (female sex Studies on female S. littoralis demonstrated a similar pheromone components and hairpencil components) pattern as H. virescens females, having ORNs and central have projections near the base of the antennal nerve (See PNs that responded to (Z)-9, (E)-11-tetradecenyl acetate, Fig. 11 for a summary of AL projections identified in the female sex pheromone (Ochieng’ et al. 1995; Sadek this study). Only ORNs within sensilla sensitive to both et al. 2002). The behavioral and evolutionary signifi- pheromonal odorants Z9-14:Ald or Z9-16:Ald projected cance for female moth sensitivity to their own odors is yet beyond the base of the antennal nerve to glomerulus 20. to be determined. Additionally, ORNs sensitive to male hairpencil odor- Few studies have successfully traced ORN projec- ants also had axonal arborizations primarily in glome- tions in female moths. Todd and Baker (1996) success- ruli 59 (16:OAc, 18:OAc) and 41 (16:OH), near the base fully stained ORNs responsive to plant volatiles and of the antennal nerve. female sex pheromone in T. ni. Ochieng’ et al. (1995) A large number of ORNs responding to Z9-14:Ald found ORNs sensitive to female sex pheromone which had arborizations associated with the cLFG. Previous projected to a glomerulus at the base of the antennal work by Galizia et al. (2000) and Skiri et al. (2004) did 215

might correspond to the physiological sensillar types described in the present study. The identification and localization of glomeruli sensitive to Z9-14:Ald and other pheromone components to the base of the antennal nerve (in a similar area to that of the male MGC) provide a condition for the development of the male MGC through sexual selection, with possible intersexual homology in pheromone-processing glomeruli. Hansson and Christensen (1999) proposed a model for MGC evolution based on inter- and intra- sexual selection pressure that drives an increase in the complexity and size of glomeruli. Such a model has been supported by research on female S. littoralis, wherein female sex-pheromone sensitive ORNs arborize in an ordinary-sized glomerulus at the base of the antennal nerve, in a location similar to the male MGC (Ochieng’ et al. 1995). Todd and Baker (1996) also found evidence of functional partitioning of the female AL based upon responses to odor blends in T. ni. ORNs in T. ni responding to the major female sex pheromone component projected to an anterior glomerulus, while those excited by floral odors projected to the posterior portion of the AL. The targeting of pheromone-sensitive ORNs to the enlarged cLFG and surrounding glomeruli in H. virescens females fits well with this model of MGC development, though the informative benefit to the female moth is yet unclear. The relative position of glomeruli sensitive to hairpencil and plant volatiles is also of considerable interest. Interglomerular connectivity and local inhibition influ- ence the spatial representation of odors in the AL (Christensen et al. 2000; Lei et al. 2002). Electrophysi- ological and behavioral studies have shown that plant volatiles may synergize male responses to pheromones in Fig. 10 Type 16 sensillum responses. a b ORN response profile Helicoverpa zea Boddie and in the tortricid Cydia po- (original spike trains). A female H. virescens type 16 sensillum monella L. (Ochieng’ et al. 2002; Yang et al. 2004). responding to a b caryophyllene; b hexane blank. c Dose response curves from ORNs in type 16 sensilla (N=2) stimulated with b Accordingly, the context of various odor combinations caryophyllene. Six second total recording time, stimulus delivery may result in contrast enhancement or inhibition. The 3·100 ms pulses mechanism of such ensemble coding and interglomerular interactions has been investigated previously using female sex pheromone responses in the male MGC not find evidence of calcium-evoked signals in the female (Christensen et al. 2000; Lei et al. 2002). H. virescens AL in response to olfactory stimulation In addition to the presence of multiple projections to using Z9-14:Ald or Z11-16:Ald. One possible reason for the cLFG of Z9-14:Ald ORNs, type 12 sensilla, housing this discrepancy is that calcium- imaging studies were an ORN sensitive to linalool, showed projections to generally limited to glomeruli located near the exposed mLFG and glomerulus 36. This may be analogous to the (usually most anterior) portion of the AL. Skiri et al. organization observed in female M. sexta, wherein (2004) recognized that the cLFG was not in focus during LFGs are innervated by ORNs sensitive to plant vola- their investigation, thereby preventing visualization of tiles, and are enantiomer selective for (+)linalool within this glomerulus’ response spectrum. Cobalt-lysine the ‘lateral LFG’ (King et al. 2000; Shields and Hilde- staining permits identification of ORN projections to brand 2001; Reisenman et al. 2004). Further, in our glomeruli beneath the AL surface. The presence of study, EAG responses to linalool were elevated in fe- ORNs sensitive to female H. virescens sex pheromone males relative to males, potentially indicative of female components is supported by a high expression of ‘male specialization for this odorant, a terpenoid produced by specific’ pheromone binding proteins on the antenna, H. virescens host species (De Moraes et al. 1999, 2001). which have been described previously in female H. vi- Unfortunately, only two successful stains were made rescens (Callahan et al. 2000). Kreiger et al. (2004) also from ORNs in this sensillar type, and it is not possible to demonstrated the expression of putative pheromone differentiate which glomerulus (mLFG or 36) is the receptor genes on female H. virescens antenna, which target of the linalool-sensitive ORN. As with other 216

Fig. 11 Summary of antennal lobe glomerular projections of physiologically characterized ORNs in female H. virescens identiﬁed from this study, shown in a anterior, and b lateral aspect. Glomeruli are numbered according to the H. virescens antennal lobe atlas (Berg et al. 2002). Dorsal D; Medial M; Anterior, A

sensillar types with projections to multiple glomeruli, Acknowledgements Special thanks to J. Celestino, K. Iceman and intracellular recording and staining of AL projection D. Kelly for colony maintenance and to C. Fogarty for valuable comments on the manuscript. We are also grateful to Dr. R. Ra neurons will aid in verifying ORN projections observed guso and Dr. J. Tumlinson for kindly providing volatile stock in the current study. solutions. We also thank S.G. Lee and Dr. T.C. Baker for advice The responses to linalool were associated with ORNs with the cobalt lysine staining technique. This research was funded in several different sensillar types. Notably, ORNs in by USDA NRICGP no. 1999 03541 and NSF IBN 9905683 to both type 12 and 13 sensilla responded to linalool (plus NJV. three other plant odorants for type 13 sensilla), with projections to different areas of the AL. In the case of pheromone reception in H. virescens and other moth References species, ORNs responding to a particular odorant will project exclusively to one glomerulus (Hansson et al. Almaas TJ, Mustaparta H (1990) Pheromone reception in tobacco 1995; Hansson and Christensen 1999). The presence of budworm moth, Heliothis virescens. J Chem Ecol 16:1331 1347 Almaas TJ, Mustaparta H (1991) Heliothis virescens: Response different destinations for ORNs within linalool-sensitive characteristics of receptor neurons in sensilla trichoidea type 1 sensillar types may be a consequence of a broader tuning and type 2. J Chem Ecol 17:953 972 of these ORNs, such that some of these ORNs are Almaas TJ, Christensen TA, Mustaparta H (1991) Chemical specialized for an unidentified ligand, but still activate in communication in heliothine moths I. Antennal receptor neu rons encode several features of intra and interspecific odorants response to linalool. Thus, various ORNs may have in the male corn earworm moth Helicoverpa zea. J Comp varying sensitivity to odors, producing an ‘across-glo- Physiol A 169:249 258 merular’ code, wherein different glomerular combina- Anderson P, Hilker M, Hansson BS, Bombosch S, Klein B, tions will be activated by specific odors and Schildknecht H (1993) Oviposition deterring components in concentrations. Therefore, for an odorant such as lin- larval frass of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae): a behavioral and electrophysiological evaluation. J alool, several glomeruli may be simultaneously activated Insect Physiol 39:129 137 permitting combinatorial odor coding to discriminate Anton S, Hansson BS (1994) Central processing of sex pheromone, such odorants. host odour, and oviposition deterrent information by inter 217

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